Ribosomes are responsible for translating RNA into proteins in all living organisms. The last ten years have shown remarkable advances in understanding the complex architecture of this large protein and ribonucleic acid structure. Despite the significant progress in determining the ribosome structure, knowledge on the biogenesis of this complex still contains many important gaps. This project will study the structure of the immediate precursor to the mature, eukaryotic, small subunit, the late pre-40S assembly intermediate. The proposed work will apply electron cryomicroscopy (cryo-EM) single particle analysis to purified pre-40S subunits to obtain a map at sub-nanometer resolution. Achievement of this high-resolution cryo-EM structure will allow access to important information necessary to understand the final sequence of ribosome maturation. An important step in the transition of the late pre-40S to a mature, functioning ribosome is the cleavage of the internally transcribed spacer 1 (ITS-1) at site D. Using the cryo-EM map, one will be able to trace the path of the rRNA through the endonuclease assembly factor responsible for its cleavage, Nob1. Furthermore, this map will show how proteins associated with regulation of this cleave process interact structurally with this site. Additionally, molecular dynamics flexible fitting will be use to model the known mature eukaryotic 40S subunit crystal structure into the obtained cryo-EM map of the pre-40S. This will allow mapping of differences between the locations of ribosomal proteins and rRNA secondary structure in the mature subunit and its precursor, thereby allowing us to obtain crucial mechanistic insights in to the final maturation process. PUBLIC HEALTH RELEVANCE: Errors in the construction of a key cellular machine, the ribosome, can cause a number of diseases, including rare forms of anemia. The proposed work will study the structure of an important intermediate step in the development of the ribosome. It is hoped that through mapping this structure, key steps of ribosome development will be better understood. This knowledge will allow us to construct the structural framework for understanding the related pathologies and for designing novel therapeutic strategies.